Antigenicity comparison of SARS‐CoV‐2 Omicron sublineages with other variants contained multiple mutations in RBD

Abstract The severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) variants, particularly those with multiple mutations in receptor‐binding domain (RBD), pose a critical challenge to the efficacy of coronavirus disease 2019 (COVID‐19) vaccines and therapeutic neutralizing monoclonal antibodies (mAbs). Omicron sublineages BA.1, BA.2, BA.3, as well as the recent emergence of C.1.2, B.1.630, B.1.640.1, and B.1.640.2, have multiple mutations in RBD and may lead to severe neutralizing antibody evasion. It is urgent to evaluate the antigenic change of the above seven variants against mAbs and sera from guinea pigs immunized with variants of concern (VOCs) (Alpha, Beta, Gamma, Delta, Omicron) and variants of interest (VOIs) (Lambda, Mu) immunogens. Only seven out of the 24 mAbs showed no reduction in neutralizing activity against BA.1, BA.2, and BA.3. However, among these seven mAbs, the neutralization activity of XGv337 and XGv338 against C.1.2, B.1.630, B.1.640.1, and B.1.640.2 were decreased. Therefore, only five neutralizing mAbs showed no significant change against these seven variants. Using VOCs and VOIs as immunogens, we found that the antigenicity of variants could be divided into three clusters, and each cluster showed similar antigenicity to different immunogens. Among them, D614G, B.1.640.1, and B.1.630 formed a cluster, C.1.2 and B.1.640.2 formed a cluster, and BA.1, BA.2, and BA.3 formed a cluster.


INTRODUTION
The coronavirus disease 2019 (COVID- 19) pandemic has been going on for more than 2 years. 1,2 According to the World Health Organization (WHO), more than 440 million confirmed cases have been reported, including more than 5.9 million deaths. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen of COVID-19, has evolved and multiple variants have emerged. 3 Based on the characteristics of the virus, such as its infectivity, the severity of the associated disease, or the impact on vaccines, therapeutics, diagnostics, etc., WHO characterized variants that posed an increased risk to global public health into three categories: variants of concern (VOC), variants of interest (VOI), and variants under monitoring (VUM). 4  VUMs change rapidly, with dynamic additions and deletions. 5 Omicron, discovered in November 2021, attracted immediate and widespread attention because it contains more than 30 mutations in its spike protein. 6,7 Since then, continued monitoring of Omicron evolution 8 revealed an increased prevalence of two sublineages, BA.1.1 and BA.2. 9,10 BA.1.1 has additional R346K mutation compared to BA.1. And in early 2022, BA.2 was becoming more common in a number of countries. By February, BA.2 had become dominant worldwide, overtaking the oncedominant BA.1. 11 There were no specific mutations for the BA.3 lineage in the spike protein. Instead, it is a combination of BA.1 and BA.2 spike protein mutations. 12 17 which defined as VUM in 2021, contain more than three mutations in receptor-binding domain (RBD). These complicated variants with multiple mutations in RBD have also attracted attention.
Preliminary data from multiple studies suggested that RBD mutations impact the neutralizing activity of monoclonal antibodies (mAbs) and vaccine immune sera. 18,19 Multiple mutation sites at RBD, especially E484K mutation superimposed other mutations, may lead to breakthrough infection. 20 At present, the majority of commercial mAbs fail to provide protection against BA.1. 21 Mean-while, studies show that BA.1 significantly evades immunity from prior infection or vaccination. 22 26 In this study, the complicated variants including Omicron sublineages (BA. 1

Antigenicity assessment of the complicated variants with multiple RBD mutations against different immunogenic sera
To evaluate the antigenicity of the complicated variants with multiple RBD mutations, we immunized guinea pigs with spike trimeric proteins of D614G, VOCs (Alpha, Beta, Gamma, Delta, Omicron), and VOIs (Lambda, Mu). Serum samples were collected 4 weeks after the third immunization. Then, eight pseudoviruses, D614G, Omicron (BA.     Figure 1 variants. Cluster analysis showed that D614G, B.1.640.1, and B.1.630 showed similar antigenicity to different immunogens ( Figure 3C). The antigenicity of C.1.2 variant against these eight groups of sera can be divided into three categories ( Figure 3B,D). The C.1.2 variant was well neutralized by Beta, Gamma, and Mu spike-immunized sera, with NT50 of 7985, 4645, and 10,680, respectively. The C.1.2 variant showed slight resistance to D614G, Alpha, Delta, and Lambda spike-immunized sera with NT50 between 2020 F I G U R E 3 Antigenicity analysis of Omicron sublineages and variants under monitoring (VUMs) against variants of concern (VOCs) and variants of interest (VOIs) spike protein immunized sera. (A) Schematic diagram of the procedures of vaccine immunization and blood collection. Nine guinea pigs were subcutaneously immunized with 100 µg of spike protein and alum adjuvant mixed, and immunized three times on D0, D14, and D28, respectively. Serum was collected 28 days after the third immunization.  Figure 3B,D). They could also be neutralized by Mu spike-immunized serum with NT50 values above 4584. The BA.1, BA.2, and BA.3 variants showed slight resistance to Beta and Gamma spike-immunized sera, especially the NT50 value of BA.2 variant against Gamma spikeimmunized sera was only 2674, and BA.3 variant against Beta spike-immunized sera was only 1441. In addition, BA.1, BA.2, and BA.3 variants were strongly resistant to D614G, Alpha, Delta, and Lambda spike-immunized sera, with NT50 values ranging from 970 to 2429. Cluster analysis showed that BA.1, BA.2, and BA.3 variants showed similar antigenicity to different immunogens ( Figure 3C).

DISCUSSION
Since the outbreak of COVID  14,16 The spike proteins of these variants have a large number of mutations, especially the RBDs have more than three mutations. There is evidence that these variants can enhance the transmissibility, fitness, infectivity of the virus, and reduce the protective efficacy of vaccines and therapeutic antibodies, which quickly raised unprecedented concerns. 6,7,29 At present, the results showed that BA.1 can largely escape vaccination, convalescent serum, and most approved mAbs. 21,22,30,31 In response to Omicron, researchers quickly screened out neutralizing mAbs and specific vaccines against BA. In this study, we first tested the neutralizing activity of 24 mAbs derived from infected individuals or vaccine immune screening against seven variants. These include not only therapeutic mAbs already on the market and those under development, but also 11 mAbs derived from memory B cells in three-dose vaccinees. Our data showed that the vast majority of mAbs' neutralizing activity against BA.1, BA.2, and BA.3 variants was reduced or even lost.
Among the 24 mAbs we tested, 22 mAbs showed consistent response against BA.1, BA.2, and BA.3, and two mAbs were inconsistent, which may be related to the individual amino acid substitution in RBD. The RBD of C.1.2, B.1.630,  B.1.640.1, and B.1.640.2 variants contain three to six mutations. Although some mAbs have reduced neutralizing activity, a considerable number of mAbs retain their neutralization activity against these variants. The approved neutralizing antibody drugs REGN10933 and REGN10987 almost lost protection against Omicron sublineages, which is consistent with previous reports. 21,34 Meanwhile, the neutralizing activity of REGN10933 against C.1.2, B.1.640.1,  and B.1.640.2 variants was decreased, and the neutralizing activity of REGN10987 against B.1.640.2 variant was decreased. It is speculated that it is related to the K417 and E484 mutations. Therefore, mAbs REGN10933 and REGN10987 are not suitable for the treatment of variants with multiple RBD mutations. The neutralizing activity of the approved mAb 196 against Omicron sublineages also decreased by more than 50-fold, suggesting the need for further development of new therapeutic mAbs. 35 In addition, mAbs JS016 36 and 9MW33 37 under development almost lost their protection against Omicron sublineages, and also had slightly decreased neutralizing activity against C.1.2, B.1.640.1, and B.1.640.2 variants. The neutralizing activity of mAb JS016 was reduced due to the N501Y mutation in the variants. The neutralizing activity of 604 against Omicron sublineages was reduced, which was consistent with the previous reports. 21 AM180 and 9G11 lost neutralizing activity to the other seven variants except for D614G, presumably due to the E484 mutation. 38 Monoclonal antibody 10D12 almost lost protection against Omicron sublineages, presumably related to the K417N mutation, which is consistent with the previous reports. 38 The neutralization protection of mAb A001 against Omicron sublineages decreased, but the EC50 values against the eight pseudoviruses tested ranged from 1.2 to 11.9 ng/ml. Among the 24 mAbs we tested, A001 showed the best neutralizing activity against the variants, suggesting that this mAb has the potential to be a neutralizing antibody drug for the treatment of COVID-19. Monoclonal antibody 9A8 showed broad-spectrum neutralizing activity, and the neutralizing activity against Omicron sublineages was increased.
Eleven mAbs isolated from memory B cells of three-dose vaccinees had different neutralizing properties. 32 The neutralizing activity of mAbs XGv296, XGv297, and XGv253 against Omicron sublineages decreased by more than 10-fold, but retained the neutralization activity against C. 1.2, B.1.630, B.1.640.1, and B.1.640.2 variants. XGv337 and XGv338 showed good neutralization protection against Omicron sublineages, but the protection against C.1.2, B.1.630, B.1.640.1, and B.1.640.2 variants was reduced by more than eight-fold, presumably related to Y449 and L452 mutations. The neutralizing activity of mAb XGv282 against BA.1 and BA.3 was decreased by 62.8-and 18.5-fold, but there was no significant change against BA.2. Structural analysis showed that the antigenic binding epitope of XGv282 was located at the right shoulder of RBD, whereas the G446S mutation of BA.1 and BA.3 affected the binding of the mAb. 32 In addition, the neutralization activity of XGv282 against C.1.2 decreased by 606.8-fold, which was speculated to be related to the Y449H mutation of C.1.2 variant. Surprisingly, the neutralizing activity of XGv282 against B.1.640.1 was reduced by 89.5-fold, but no significant change against B.1.640.2, presumably due to the additional E484K mutation altering the spatial conformation of the spike protein. The neutralizing activity of XGv052 against BA.1 variant was reduced by 6.4fold, but the neutralizing activity against BA.2 and BA.3 did not change. It was speculated that the BA.1 variant RBD contained G446S and G496S, whereas the BA.2 and BA.3 variants did not contain these two mutations, thus leading to this result. The mAbs XGv264, XGv286, XGv293, and XGv347 exhibited broad-spectrum neutralizing activity against the eight pseudoviruses tested. Surprisingly, the neutralizing activity of XGv347 against Omicron sublineages was not reduced, but enhanced. In previous literature, its structure was analyzed and it was found that the XGv347-Omicron S complex structures had three distinct conformational states. Moreover, XGv347 binds to an epitope at RBD, largely overlapping with the patch targeted by ACE2. And this epitope is conserved among different SARS-CoV-2 variants, resulting in the broad-spectrum of XGv347 to VOCs and VUMs.
In this study, the recombinant spike trimer protein was used as an immunogen to simulate vaccination or natural infection. The antigenicity analysis of variants showed that the variants could be divided into three clusters, and each cluster showed similar antigenicity to different immunogens. Among them, D614G, B.1.640.1, and B.1.630 formed a cluster, C.1.2 and B.1.640.2 formed a cluster, and BA.1, BA.2, and BA.3 formed a cluster. However, it has been documented that although BA.1 and BA.2 both evaded the vaccine-induced antibody response, this phenomenon resulted from different antigenic characteristics. 39 Thus, it is speculated that Omicron BA.1 and BA.2 are antigenically distinct SARS-CoV-2 variants. 39 But there is no doubt that both the immunogenicity and antigenicity of Omicron have evolved into a relatively distant branch compared to other variants. This is consistent with clinical manifestations of Omicron in the real world. It has been reported that after immunization with inactivated vaccine, recombinant protein vaccine and mRNA vaccine, the antibody level decreases with the extension of inoculation time, leading to the increase of breakthrough infection of Omicron. 18,22 Our data indicated that D614G, Alpha, Delta, and Mu were similar in immunogenicity, and these sera had significantly reduced protection against Omicron sublineages ( Figure S1). Surprisingly, these four groups of sera also had poor neutralizing activity against C.1.2 and B.1.640.2, which may be caused by E484K mutation. The strong neutralization protection of Delta spike-immunized sera against B.1.630 may be due to the L452R mutation contained in the Delta immunogen. Similarly, the strong neutralization protection of Lambda spike-immunized sera against B.1.630 and B.1.640.1 may be due to the presence of L452Q (B.1.630 containing L452R mutation) and F490S (B.1.640.1 containing F490R mutation but not E484K mutation) in the Lambda immunogen. The immunogenicity of Beta, Gamma, and Mu were similar, probably because these three immunogens all contained E484K and N501Y mutations ( Figure S1). Surprisingly, these immunogens exhibited protection against Omicron. Before the outbreak of Omicron, several vaccine companies had developed a new generation of vaccines. 40 The mRNA vaccine and recombinant protein vaccine against the Beta variant are already in the clinical studies. 41,42 Therefore, it is speculated that the Beta vaccine booster can achieve partial resistance to the Omicron variants. Additionally, the Mu immunogen can induce high neutralizing protective antibodies against a broad spectrum of variants, which is also worthy of further study ( Figure S1). Besides, we found significant differences in immunogenicity between Omicron and these seven variants. Omicron spike-immunized sera can well neutralize the BA.1, BA.2, BA.3 variants ( Figure S1). This is consistent with the results of Omicronspecific mRNA vaccine, the antibodies produced by the Omicron vaccine immunization can achieve protection against Omicron variants. 33,43 In addition, Omicron spikeimmunized sera also has a certain protective effect on B.1.630, possibly due to Omicron immunogen containing T478K and E484A. However, Omicron-immunized sera was extremely poorly protected against the other variants. This suggests that natural infection with Omicron and specific vaccines developed against Omicron may not provide useful or broad herd immunity against other variants. Therefore, the Omicron-specific vaccine can be used as a booster vaccine, but it cannot replace the original vaccine.
The limitation of this study is that we only used pseudotyped viruses and did not use live viruses for validation. However, it is worth noting that pseudoviruses are now widely used in virus research and vaccine evaluation, and have been shown to be well correlated with authentic viruses. 39 At the same time, most of the mAbs we tested are still in preclinical studies, and only a few mAbs have been used in clinical practice. In addition, in order to compare the differences between Omicron sublineages, we selected some mAbs that are known to protect the BA.1 variant, which may partially conceal the fact that Omicron has a huge challenge to existing mAbs. Besides, we only used sera from guinea pigs and did not test sera from humans. This is because it is difficult to obtain human sera cohorts immunized or infected with VOCs and VOIs variants in the same population background, at the same dose, and at the same time interval. Therefore, in order to accurately compare cross-immune responses between all significant variants, animal experiments are the fastest and optimal option.
Taken together, our study shows that a few mAbs have strong and broad-spectrum neutralizing activity against the tested variants. People vaccinated with the original strain or infected with Alpha, Delta, and Lambda may not resist to Omicron infection. At the same time, Omicronelicited antibodies were also insufficient to protect against the other variants. In addition, there were differences in the antigenicity of variants. These antigenicity differences were mainly caused by mutations in the RBD. These results suggest that E484, N501, and other sites may be important to be considered in the development of the next-generation vaccines.

Monoclonal antibodies
Twenty-four anti-SARS-CoV-2 spike mAbs were used in this study. The mAb sources were as follows: mAbs A001

Sera from guinea pigs immunized with SARS-CoV-2 variant spike protein
Female guinea pigs (bodyweight 200-220 g) were used as experimental animals, and divided into eight groups with nine animals in each group. Each guinea pig was subcutaneously immunized with 100 µg of purified spike proteins of different SARS-CoV-2 variants (Acro Biosystems Co.), including the D614G reference strain, current VOCs (Alpha, Beta, Gamma, Delta, Omicron) and VOIs (Lambda, Mu). Spike protein (100 µg) was mixed with alum adjuvant and immunized once every 14 days for three inoculations in total. Sera were collected 28 days after the third immunization for subsequent experiments.

Construction and titration of SARS-CoV-2 pseudotyped viruses
The pseudotyped viruses bearing the spike protein were generated and titrated as previously described. Briefly, 293T cells were first transfected with the SARS-CoV-2 spike protein expression plasmids of D614G or VOCs/VOIs (Alpha, Beta, Gamma, Delta, Lambda, Mu, and Omicron). The transfected 293T cells were simultaneously infected with VSV pseudotyped virus, G*ΔG-VSV (Kerafast, Boston, MA). After 6 h of incubation, cells were washed twice with PBS before complete culture medium was added. Pseudovirus-containing supernatants were harvested after 24 and 48 h, and stored at −80 • C for future use. Titrations of SARS-CoV-2 pseudotyped viruses were assessed by infecting Huh-7 cells with three-fold serial dilutions. The cell culture plate was incubated at 37 • C with 5% CO 2 for 24 h. Chemiluminescent signals were detected according to the protocol of the Britelite plus reporter gene assay system (PerkinElmer, Waltham, MA).

4.6
In vitro neutralization assay with pseudotyped viruses For the in vitro pseudotyped virus neutralization assays, plasma samples (starting at 1:30) or antibodies (appropriate concentrations) were serially diluted and mixed with 1.3 × 10 4 TCID 50 of pseudotyped viruses in 96-well plates at 37 • C for 1 h. It was then mixed with Huh-7 cells (20,000/wells) and subsequently incubated for 24 h. As previously described, the luciferase luminescence (RLU) of each well was measured, and the 50% neutralization titer (NT50) was calculated using the Reed-Muench method to evaluate the neutralizing antibody content in the samples.

Spearman's correlation coefficient heatmap
The NT50 values corresponding to each serum/virus were log scale transformed, assembled into an 8 × 8 matrix. A matrix of Spearman's correlation coefficients (r 2 ) between each virus strain is displayed in the form of a heatmap.

Software and data analysis
Figures were generated using GraphPad Prism 8 software (GraphPad, San Diego, CA, USA) and Microsoft Excel. Values are shown as means with range. The least squares fit of Abs is calculated by sigmoidal 4PL (where X is log concentration) standard curve. Spearman's correlation coefficient (r 2 ) heatmap of virus strain was generated using Python language (https://www.python.org).

A C K N O W L E D G M E N T S
We thank GISAID for viral sequence information. We thank Acro Biosystems Co. for providing the recombinant spike proteins of D614G, Alpha, Beta, Gamma, Delta, Lambda, Mu, and Omicron. We thank BioRender.com for providing drawing materials.

C O N F L I C T O F I N T E R E S T Qianqian Li is an employee of Jiangsu Recbio Technology
Co., Ltd. All other authors declare that there is no conflict of interest.

E T H I C S A P P R O VA L
Guinea pigs were handled under institutional (NIFDC, Beijing, China) guidelines for laboratory animal care and use, and the animal study protocol was approved by the Animal Care and Use Committee of the NIFDC.

D ATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analyzed during this study are available from the corresponding author upon reasonable request.